The ability to introduce specific alterations of endogenous genes into the germline of mice via targeted mutagenesis in embryonic stem cells (ES) has represented a major breakthrough in mouse genetics. Thus gene inactivation has been widely used to examine the effect of loss of function in various biological processes and has already permitted to accumulate a wealth of new insights into gene function and also to create mouse models of human genetic diseases. However it would also be useful to introduce subtle mutations, in order to refine the genetic analysis and to better approximate the models of genetic diseases which do not necessarily result from null mutations. Thus several strategies have been developped, aimed at generating subtle mutations in a given gene. However, one common limitation to all the current gene targeting procedures is the low frequency of correct targeting, which becomes a serious problems especially when using two successive rounds of targeting. Therefore, efforts have been made to increase the efficiency of gene targeting by several means like increasing the size of the region of homologies with the target locus, using isogenic genomic DNA, or improving the selection procedures.
In this paper, we present an alternative approach, to overcome these limitations, which relies on the observation that double-strand ends of broken chromosomes are highly recombinogenic; we reasoned that the introduction of a double stranded break (DSB) in an endogenous gene could increase targeting frequency through stimulation of the cellular recombination machinery. Thus our approach which involves two steps, is based on the induction of a specific DSB in a natural locus, the gene encoding villin, an actin binding protein mainly expressed in the intestine and kidney (louvard). In a first step, an I-Sce I restriction site is introduced into the villin locus. Indeed, I-Sce I, a very rare cutter endonuclease has been shown to initiate DSB in the mammalian genome, while its random integration allowed new insights into the analysis of recombination mechanisms in mammals. In the second step, the effect of DSB on DNA repair and homologous recombination frequency is assessed after cotransfer of an I-Sce I expressing vector and of a villin replacement vector in the targeted ES cells (FIG. 1). We found that the presence of a DSB in the target gene not only enhanced homologous replacement by the incoming DNA but also induced gene conversion at a very high rate.